Granule With Hydrated Barrier Material

ABSTRACT

A granule having high stability and low dust is described. The granule includes a hydrated barrier material having moderate or high water activity. Also described are methods of producing the granules.

BACKGROUND OF THE INVENTION

Recently the use of enzymes, especially of microbial origin, has becomemore and more common. Enzymes are used in several industries including,for example, the starch industry, the dairy industry, and the detergentindustry. It is well known in the detergent industry that the use ofenzymes, particularly proteolytic enzymes, has created industrialhygiene concerns for detergent factory workers, particularly due to thehealth risks associated with dustiness of the available enzymes.

Since the introduction of enzymes into the detergent business, manydevelopments in the granulation and coating of enzymes have been offeredby the industry. See for example the following patents relating toenzyme granulation:

U.S. Pat. No. 4,106,991 describes an improved formation of enzymegranules by including within the composition undergoing granulation,finely divided cellulose fibers in an amount of 2-40% w/w based on thedry weight of the whole composition. In addition, this patent describesthat waxy substances can be used to coat the particles of the granulate.

U.S. Patent 4,689,297 describes enzyme containing particles whichcomprise a particulate, water dispersible core which is 150-2,000microns in its longest dimension, a uniform layer of enzyme around thecore particle which amounts to 10%-35% by weight of the weight of thecore particle, and a layer of macro-molecular, film-forming, watersoluble or dispersible coating agent uniformly surrounding the enzymelayer wherein the combination of enzyme and coating agent is from 25-55%of the weight of the core particle. The core material described in thispatent includes clay, a sugar crystal enclosed in layers of corn starchwhich is coated with a layer of dextrin, agglomerated potato starch,particulate salt, agglomerated trisodium citrate, pan crystallized NaClflakes, bentonite granules or prills, granules containing bentonite,Kaolin and diatomaceous earth or sodium citrate crystals. The filmforming material may be a fatty acid ester, an alkoxylated alcohol, apolyvinyl alcohol or an ethoxylated alkylphenol.

U.S. Pat. No. 4,740,469 describes an enzyme granular compositionconsisting essentially of from 1-35% by weight of an enzyme and from0.5-30% by weight of a synthetic fibrous material having an averagelength of from 100-500 micron and a fineness in the range of from0.05-0.7 denier, with the balance being an extender or filler. Thegranular composition may further comprise a molten waxy material, suchas polyethylene glycol, and optionally a colorant such as titaniumdioxide.

U.S. Pat. No. 5,254,283 describes a particulate material which has beencoated with a continuous layer of a non-water soluble, warp sizepolymer. U.S. Pat. No. 5,324,649 describes enzyme-containing granuleshaving a core, an enzyme layer and an outer coating layer. The enzymelayer and, optionally, the core and outer coating layer contain a vinylpolymer.

WO 91/09941 describes an enzyme containing preparation whereby at least50% of the enzymatic activity is present in the preparation as enzymecrystals. The preparation can be either a slurry or a granulate.

WO 97/12958 discloses a microgranular enzyme composition. The granulesare made by fluid-bed agglomeration which results in granules withnumerous carrier or seed particles coated with enzyme and bound togetherby a binder.

However, even in light of these developments offered by the industry (asdescribed above) there is a continuing need for low-dust enzyme granuleswhich have additional beneficial characteristics. Additional beneficialcharacteristics needed in the enzyme granulation industry arelow-residue granule formulations (where low residue is defined as areduced tendency to leave noticeable undissolved residues on clothes orother material), and improved stability formulations. Accomplishing allthese desired characteristics simultaneously is a particularlychallenging task since, for example, many delayed release or low-dustagents such as fibrous cellulose or warp size polymers leave behindinsoluble residues.

Therefore, it is an object of the present invention to provide low-dust,low residue, highly soluble enzyme granules having increased stability.It is another object of the present invention to provide processes whichafford the formation of such improved granules.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a granule that includes aprotein core and a hydrated barrier material with moderate or high wateractivity. The hydrated barrier material can be in one or more layersand/or can be included in the protein core.

A further embodiment of the present invention is a granule that includesan enzyme core and a hydrated barrier material with moderate or highwater activity. The hydrated barrier material can be in one or morelayers and/or can be included in the enzyme core.

Another embodiment is a method of producing the above granule.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a granule with improved stability havinglow dust. The granule includes a protein core and a hydrated barriermaterial with moderate or high water activity.

A “protein core” or an “enzyme core” can be homogenous such as thatdescribed in U.S. patent application Ser. No. 08/995,457 or layered asdescribed in U. S. Pat. No. 5,324,649.

Proteins that are within the scope of the present invention includepharmaceutically important proteins such as hormones or othertherapeutic proteins and industrially important proteins such asenzymes.

Any enzyme or combination of enzymes may be used in the presentinvention. Preferred enzymes include those enzymes capable ofhydrolyzing substrates, e.g. stains. These enzymes are known ashydrolases which include, but are not limited to, proteases (bacterial,fungal, acid, neutral or alkaline), amylases (alpha or beta), lipases,cellulases and mixtures thereof. Particularly preferred enzymes aresubtilisins and cellulases. Most preferred are subtilisins such asdescribed in U.S. Pat. No. 4,760,025, EP Patent 130 756 B1 and EP PatentApplication WO 91/06637, which are incorporated herein by reference, andcellulases such as Multifect L250™ and Puradax™, commercially availablefrom Genencor International. Other enzymes that can be used in thepresent invention include oxidases, transferases, dehydratases,reductases, hemicellulases and isomerases.

As noted, the barrier material can be coated over the protein core inone or more layers or made part of the protein core in order to insulateor to impede transport of water and inactivating substances to theprotein. When the barrier material is part of the protein core, it canbe dispersed throughout the core or as a layer in the core.

Suitable hydrated barrier materials with moderate or high water activitycan include salts of an inorganic or organic acid, sugars,polysaccharides, lipids, proteins or synthetic polymers; preferablysalts.

The term “water activity”, symbolized a_(w), refers to the fractionalrelative humidity of an atmosphere in equilibrium with a solid or liquidphase material, i.e., the ratio of the partial pressure of water vaporto that present above pure water at the same temperature. In all phasesbetween which water distribution has reached equilibrium, it is bydefinition equal. The term “relative humidity” is generally used todescribe the water in the atmosphere or gas phase in equilibrium withthe solid, and is expressed as a percentage, with 100% as the relativehumidity of pure water in a closed system. Thus, for any water activityvalue, there is a corresponding relative humidity given by %RH=100*a_(w).

Water activity can be readily measured by methods known in the art,typically by placing a sample of the material inside thetemperature-controlled chamber of a water activity meter, such as theWater Activity System Model D2100 available from Rotronic InstrumentCorp. (Huntington, N.Y.), and allowing the measurement to reachequilibrium as indicated on the display.

A “hydrated” barrier material contains water in a free or bound form, ora combination of the two. The water of hydration can be added eitherduring or after the coating process. The degree of hydration will be afunction of the material itself and the temperature, humidity and dryingconditions under which it is applied.

“Moderate or high” water activity includes a water activity of at least0.25, preferably greater than 0.30, most preferably greater than 0.35.The water activity referred to herein is that of the granule itself onceit has the barrier material—but no further coatings—coated onto it.Further coatings may mask accurate measurement of the water activity ofthe barrier material as a distinct layer.

Without wishing to be bound by theory, it is expected that materialswith a water activity greater than 0.25 will have a reduced drivingforce for picking up water under storage conditions in which therelative humidity is greater than 25%. Most climates have relativehumidities above 25%. Many detergents have water activities in the rangeof about 0.3 to 0.4. If the water activity of the granule is actuallyhigher than that of the surrounding detergent or storage climate, thedriving force for pick up of water by the granule should be eliminated,and in fact water may be given up by the granule to its surroundings.Even if the water activity of the granule is lower than that of thedetergent or the corresponding relative humidity, the water present inthe barrier layer would act as a shield limiting the amount of water andhence in activating substances being picked up by the granule andaffecting the protein core.

In the case of salt hydrates, the hydrated material is a crystallinesalt hydrate with bound water(s) of crystallization. The hydrate shouldbe chosen and applied in a manner such that the resulting coated granulewill have a water activity in excess of 0.25, or as high as possiblewhile still providing a granule which is dry to the touch. By applying asalt hydrate, or any other suitable hydrated barrier material, in such amanner, as noted above, one expects that this would eliminate anydriving force for further uptake of water by the granule. As animportant consequence, the driving force for transport of substanceswhich may be detrimental to enzyme activity, such as perborate orperoxide anion, is removed. Without water as a vehicle, these substancesare less likely to penetrate the enzyme core. Empirical datademonstrates that enzyme activity in the granule is substantiallyenhanced by coating the enzyme core with stable salt hydrates.

Preferred salts include magnesium sulfate heptahydrate, zinc sulfateheptahydrate, copper sulfate pentahydrate, sodium phosphate dibasicheptahydrate, magnesium nitrate hexahydrate, sodium borate decahydrate,sodium citrate dihydrate and magnesium acetate tetrahydrate.

The granules of the present invention can also comprise one or morecoating layers. For example, such coating layers may be one or moreintermediate coating layers, or such coating layers may be one or moreoutside coating layers or a combination thereof. Coating layers mayserve any of a number of functions in a granule composition, dependingon the end use of the granule. For example, coatings may render theprotein resistant to oxidation by bleach, bring about the desirablerates of dissolution upon introduction of the granule into an aqueousmedium, or provide a barrier against ambient moisture in order toenhance the storage stability of the enzyme and reduce the possibilityof microbial growth within the granule.

Suitable coatings include polyvinyl alcohol (PVA), polyvinyl pyrrolidone(PVP), cellulose derivatives such as methylcellulose,hydroxypropylmethyl cellulose, hydroxycellulose, ethylcellulose,carboxymethyl cellulose, hydroxypropyl cellulose, polyethylene glycol,polyethylene oxide, chitosan, gum arabic, xanthan, carrageenan, latexpolymers, and enteric coatings. Furthermore, coating agents may be usedin conjunction with other active agents of the same or differentcategories.

Suitable PVAs for incorporation in the coating layer(s) of the granuleinclude partially hydrolyzed, fully hydrolyzed and intermediatelyhydrolyzed PVAs having low to high degrees of viscosity. Preferably, theouter coating layer comprises partially hydrolyzed PVA having lowviscosity. Other vinyl polymers which may be useful include polyvinylacetate and polyvinyl pyrrolidone. Useful copolymers include, forexample, PVA-methylmethacrylate copolymer and PVP-PVA copolymer.

The coating layers of the present invention may further comprise one ormore of the following: plasticizers, extenders, lubricants, pigments,and optionally additional enzymes. Suitable plasticizers useful in thecoating layers of the present invention are plasticizers including, forexample, polyols such as sugars, sugar alcohols, or polyethylene glycols(PEGs), urea, glycol, propylene glycol or other known plasticizers suchas triethyl citrate, dibutyl or dimethyl phthalate or water. Suitablepigments useful in the coating layers of the present invention include,but are not limited to, finely divided whiteners such as titaniumdioxide or calcium carbonate or colored pigments or dyes or acombination thereof. Preferably such pigments are low residue pigmentsupon dissolution. Suitable extenders include sugars such as sucrose orstarch hydrolysates such as maltodextrin and corn syrup solids, clayssuch as kaolin and bentonite, and talc. Suitable lubricants includenonionic surfactants such as Neodol, tallow alcohols, fatty acids, fattyacid salts such as magnesium stearate and fatty acid esters.

The outer coating layer of the present invention preferably comprisesbetween about 1-25% by weight of the coated granule.

Adjunct ingredients may be added to the granules of the presentinvention. Adjunct ingredients may include: metallic salts;solubilizers; activators; antioxidants; dyes; inhibitors; binders;fragrances; enzyme protecting agents/scavengers such as ammoniumsulfate, ammonium citrate, urea, guanidine hydrochloride, guanidinecarbonate, guanidine sulfamate, thiourea dioxide, monoethanolamine,diethanolamine, triethanolamine, amino acids such as glycine, sodiumglutamate and the like, proteins such as bovine serum albumin, caseinand the like etc.; surfactants including anionic surfactants, ampholyticsurfactants, nonionic surfactants, cationic surfactants and long-chainfatty acid salts; builders; alkalis or inorganic electrolytes; bleachingagents; bluing agents and fluorescent dyes and whiteners; and cakinginhibitors.

The granules described herein may be made by methods known to thoseskilled in the art of enzyme granulation, including pan-coating,fluid-bed coating, fluid-bed agglomeration, prilling, disc granulation,spray drying, extrusion, centrifugal extrusion, spheronization, drumgranulation, high shear agglomeration, or combinations of thesetechniques.

The following examples are representative and not intended to belimiting. One skilled in the art could choose other proteins, proteincores, enzymes, enzyme cores, seed particles, methods and coating agentsbased on the teachings herein.

EXAMPLES Example 1

Stability of Magnesium Sulfate Coated Protease Granules

A. In a Deseret 60 fluidized bed coater, 54.1 kg of sucrose/starch nonpareil seeds were charged and fluidized. Onto these cores, 75.8 kg ofprotease UF concentrate containing 62.9 g/kg subtilisin protease weresprayed under the following conditions. (Ranges indicate initial andfinal values over the course of the specified ramp time):

Ramp time: 80 minutes Fluid feed rate 0.6-1.0 liter/min Atomizationpressure 75 psi Inlet air temperature 85-92 degrees C. Outlet airtemperature 50 degrees C. Fluidization air rate 18 m3/min

A solution of magnesium sulfate was prepared by adding 22.2 kg ofmagnesium sulfate heptahydrate into 22.2 kg of water, and this wassprayed onto the enzyme-coated cores under the following conditions inorder to provide that 20% of the final granule would be magnesiumsulfate heptahydrate, with care being taken to keep the bed temperatureclose to, but slightly below, 50 degrees C.:

Ramp time: 40 minutes Fluid feed rate 0.6-1.7 liter/min Atomizationpressure 45 psi Inlet air temperature 70-84 degrees C. Outlet airtemperature 48-50 degrees C. Fluidization air rate 18 m3/min

Finally, a polymer coating solution was prepared by dissolving 6.35 kgof Elvanol 51-05 polyvinyl alcohol, 7.94 kg titanium dioxide and 1.59 kgNeodol 23-6.5T nonionic surfactant in 50.12 kg water and spraying overthe salt-coated enzyme cores under the following conditions:

Ramp time: 10 min, then constant for 100 min Fluid feed rate 0.6liter/min Atomization pressure 75 psi Inlet air temperature 50 degreesC. Outlet air temperature 75-80 degrees C. Fluidization air rate 18m3/min

The harvested granules had an enzyme concentration of approximately 40g/kg.

B. Accelerated Stability Test

The stability of many enzyme granules formulated into bleach-containingdetergents is generally excellent, showing generally no more than about10 to 20% loss in activity over 6 weeks storage at 30 to 37° C. and 70%to 80% R.H. However, to aid in the development and screening of granularformulations, it is desirable to have an accelerated means ofdetermining relative granule stability. The conditions of theaccelerated stability test (AST) are far more severe than enzymegranules or detergents would ever encounter in realistic storage ortransport. The AST is a “stress test” designed to discriminatedifferences between formulations which would otherwise not be evidentfor weeks or months.

In this test, a test detergent base was made from the followingingredients:

72% WFK-1 detergent base (WFK, Forschunginstitut fuerReinigungstechnologie e.V., Krefeld, Germany) 25% sodium perboratemonohydrate (Degussa Corp., Allendale Park, New Jersey.) 3% TAED bleachactivator (Warwick International, (=tetraacetylethylenediamine) Mostyn,UK)

For each enzyme sample to be tested, three identical tubes were preparedby adding 1 gram of the test base and 30 mg of enzyme granules to a 15ml conical tube and mixed by inverting the capped tube 5-8 times byhand. A hole was drilled in the tube cap with a 1/16 inch drill bit. Oneof the three tubes was assayed immediately and the other two were storedin a humidity chamber set at 50° C. and 70% R.H. One of the two storedtubes was assayed after 1 day of storage; the second, after 3 days ofstorage. Storage stability was reported for Day 1 and Day 3 by dividingthe remaining activity by the original activity at Day 0, expressed as apercentage.

The enzyme activity was determined by adding to each tube 30 ml of 0.25MMES pH 5.5 buffer containing 20 μl Catalase HP L5000 (GenencorInternational, Rochester, N.Y.) and incubating for 40 minutes toinactivate the perborate. After this, the enzyme was assayed by adding10 μl of the test tube mixture and 10 μl of sAAPF protease substrate to980 μl of 0.1 M Tris pH 8.6, then incubating at 25° C. over 3 minutes,and measuring the optical absorbance at 410 nm. The slope of theabsorbance vs. time was then multiplied by the dilution factor and theknown extinction coefficient for the specific protease to obtain anenzyme activity as concentration in mg/ml.

The process described in A above was repeated three more times, the onlydifference being that the outlet air temperature was controlled at asetpoint of 40, 60 and 70 degrees C. in each of the three separate runs.Samples were removed from all four batches after the magnesium sulfatebarrier coating had been applied, and water activities of the granuleswere measure in a Rotronic Water Activity System, as reported inTable 1. Two of the granules, after application of the final polymercoating, were placed in WFK-1 detergent formula and stored in tubes withdrilled caps for three days at 50 degrees C. and 70% relative humidity,according to the accelerated stability test method described above.Tubes were withdrawn from the humidity chamber and assayed after 1 dayand 3 days. The percent retained activities are reported in Table 1. Theresults indicate the granules in which magnesium sulfate was coated at50 degrees C. outlet temperature are significantly more stable thanthose coated at 70 degrees C., and that the more stable granules had awater activity above 0.35, while the less stable granules had asignificantly lower water activity.

TABLE 1 Stability of Magnesium Sulfate Coated Enzyme Granules A_(w) ofMgSO4 Percent Retained Activity Outlet Coated of Granules Stored TempProtease in Bleach Detergent (C.) Cores 0 days 1 day 3 days 40 0.374 500.409 100% 108% 97% 60 0.140 70 0.165 100% 94% 63%

Example 2

Stability of Sodium Citrate Coated Protease Granules

A. In a Vector 60 coater, 25 kg of sucrose/starch nonpareil seeds werefluidized and 30.9 kg of subtilisin protease concentrate with aconcentration of 65.9 g/L and 18.3% total solids were sprayed onto thefluidized cores under the following conditions:

Ramp time: 55 minutes Fluid feed rate 0.5-0.9 liter/min Atomizationpressure 75 psi Inlet air temperature 60-95 degrees C. Outlet airtemperature 50 degrees C. Fluidization air rate 24 m3/min

A solution of trisodium citrate was prepared by adding 13.2 kg oftrisodium citrate dihydrate into 19.7 kg of water, and this was sprayedonto the enzyme-coated cores under the following conditions in order toprovide that 25% of the final granule would be trisodium citratedihydrate, with care being taken to keep the bed temperature close to 50degrees C.:

Ramp time: 23 minutes Fluid feed rate 0.6-1.9 liter/min Atomizationpressure 75 psi Inlet air temperature 60-95 degrees C. Outlet airtemperature 50 degrees C. Fluidization air rate 24 m3/min

Finally, a polymer coating solution was prepared by dissolving 2.94 kgMethocel HPMC, 0.98 kg polyethylene glycol, molecular weight 600, 2.06kg titanium dioxide and 0.59 kg Neodol 23-6.5T nonionic surfactant in55.88 kg water and spraying over the salt-coated enzyme cores under thefollowing conditions:

Ramp time: 10 min, then 80 minutes constant Fluid feed rate 0.5-0.7liter/min Atomization pressure 75 psi Inlet air temperature 75-80degrees C. Outlet air temperature 60 degrees C. Fluidization air rate 18m3/min

The harvested granules had a weight of 49.5 kg and an enzymeconcentration of approximately 40 g/kg.

B. The above process was repeated under the same conditions, but theoutlet air temperature was controlled at a setpoint of 70 degrees C.Samples were removed from both batches after the sodium citrate barriercoating had been applied, and water activities of the granules weremeasure in a Rotronic Water Activity System, as reported in Table 2. Thetwo granules, after application of the final polymer coating, wereplaced in an automatic dish detergent base and stored in sealed tubesfor 84 days at 37 degrees C. Tubes were withdrawn from the humiditychamber and assayed after 14, 42 and 84 days. The percent retainedactivities are reported in Table 2. The results indicate the granules inwhich sodium citrate was coated at 50 degrees C. outlet temperature aresignificantly more stable than those coated at 70 degrees C., and thatthe more stable granules had a water activity above 0.25, while the lessstable granules had a significantly lower water activity.

TABLE 2 Stability of Sodium Citrate Coated Enzyme Granules A_(w) of Na3Citrate Outlet Coated Percent Retained Activity of Temp ProteaseGranules Stored in Bleach Detergent (C.) Cores 0 days 14 days 42 days 84days 55 0.272 100% 90% 89% 87% 70 0.059 100% 86% 81% 75%

1-11. (canceled)
 12. A granule comprising an enzyme core, a hydratedbarrier salt coated over the enzyme core, and one or more coating layerscoated over the hydrated barrier salt, wherein the hydrated barrier saltis magnesium sulfate heptahydrate.
 13. A method of producing the granuleaccording to claim 12 comprising: a) providing the enzyme core; b)coating the hydrated barrier salt onto the enzyme core; and c) applyingthe one or more coating layers over the hydrated barrier salt.